It is widely suspected that gene regulatory networks are highly plastic. The rapid turnover of transcription factor binding sites has been predicted on theoretical grounds and has been experimentally demonstrated in closely related species. We combined experimental approaches with comparative genomics to focus on the role of combinatorial control in the evolution of a large transcriptional circuit in the fungal lineage. Our study centers on Mcm1, a transcriptional regulator that, in combination with five cofactors, binds roughly 4% of the genes in Saccharomyces cerevisiae and regulates processes ranging from the cell-cycle to mating. In Kluyveromyces lactis and Candida albicans, two other hemiascomycetes, we find that the Mcm1 combinatorial circuits are substantially different. This massive rewiring of the Mcm1 circuitry has involved both substantial gain and loss of targets in ancient combinatorial circuits as well as the formation of new combinatorial interactions. We have dissected the gains and losses on the global level into subsets of functionally and temporally related changes. One particularly dramatic change is the acquisition of Mcm1 binding sites in close proximity to Rap1 binding sites at 70 ribosomal protein genes in the K. lactis lineage. Another intriguing and very recent gain occurs in the C. albicans lineage, where Mcm1 is found to bind in combination with the regulator Wor1 at many genes that function in processes associated with adaptation to the human host, including the white-opaque epigenetic switch. The large turnover of Mcm1 binding sites and the evolution of new Mcm1–cofactor interactions illuminate in sharp detail the rapid evolution of combinatorial transcription networks.
Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents. We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest. Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entrée into some forms of genomic instability. Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants. This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest. Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.
Author(s): Galgoczy, David J | Advisor(s): Johnson, Alexander D | Abstract: The coordination of cellular processes is largely controlled at the level of transcriptional regulation. Previous work, dating back over forty years, has focused on a few details of transcriptional regulation, providing a window through which to view the basic workings of transcriptional regulation. Recent developments have allowed broader analyses, providing new views into how whole transcriptional programs mold cellular processes.To provide the first complete example of a transcriptional circuit, we completely dissected the transcriptional regulation that underlies the specification of the three cell types in Saccharomyces cerevisiae. In addition to providing an example of a completely mapped circuit, this analysis uncovered genes not expected to be associated with specific cell types. We have extended this approach to a second fungal species, uncovering processes of molecular evolution on a whole circuit level.We adopted a similar approach to describe the transcriptional regulatory mechanism that governs the switch between the white and opaque states in Candida albicans, the primary fungal pathogen in humans. This process modulates the virulence properties and mating competence of this species, and involves the regulation of hundreds of genes. We showed that one transcriptional regulator, Wor1 governs the mechanism underlying the switch. Our analysis revealed that Wor1 directs an extensive and complex transcriptional program to set up the opaque state.Finally, to understand how changes in transcriptional regulation shape evolution of organisms, we explored the evolution of a large transcriptional circuit in fungi. This is an important endeavor, since phenotypic differences between evolutionarily divergent species are thought to be largely due to differences in gene expression. While several anecdotal examples of molecular evolution events exist, a broad analysis would yield insights into what types of changes figure importantly in shaping evolution on a systems level. To this end, we mapped the large combinatorial circuit controlled by Mcm1 in three fungal species; this provided an unprecedented view into the evolution of a large transcriptional circuit, and offered two examples where circuitry had been dramatically restructured. Ongoing informatics analysis is refining this view, allowing us to distill out the molecular processes that are important in the evolution of circuits.
White–opaque switching in the human fungal pathogen Candida albicans is an alternation between two distinct types of cells, white and opaque. White and opaque cells differ in their appearance under the microscope, the genes they express, their mating behaviors, and the host tissues for which they are best suited. Each state is heritable for many generations, and switching between states occurs stochastically, at low frequency. In this article, we identify a master regulator of white–opaque switching (Wor1), and we show that this protein is a transcriptional regulator that is needed to both establish and maintain the opaque state. We show that in opaque cells, Wor1 forms a positive feedback loop: It binds its own DNA regulatory region and activates its own transcription leading to the accumulation of high levels of Wor1. We further show that this feedback loop is self-sustaining: Once activated, it persists for many generations. We propose that this Wor1 feedback loop accounts, at least in part, for the heritability of the opaque state. In contrast, white cells (and their descendents) lack appreciable levels of Wor1, and the feedback loop remains inactive. Thus, this simple model can account for both the heritability of the white and opaque states and the stochastic nature of the switching between them.
The budding yeast Saccharomyces cerevisiae has three cell types (a cells, α cells, and a/α cells), each of which is specified by a unique combination of transcriptional regulators. This transcriptional circuit has served as an important model for understanding basic features of the combinatorial control of transcription and the specification of cell type. Here, using genome-wide chromatin immunoprecipitation, transcriptional profiling, and phylogenetic comparisons, we describe the complete cell-type-specification circuit for S . cerevisiae . We believe this work represents a complete description of cell-type specification in a eukaryote.
The human pathogen Candida albicans can assume either of two distinct cell types, designated "white" and "opaque." Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions.
